The budding yeast Saccharomyces cerevisiae is a microorganism with high fermentation ability and high ethanol resistance, and it has long been used to generate ethanol mainly for production of alcoholic beverages, while also being utilized in recent years for fuel ethanol production. Moreover, ethanol is also known to be a renewable form of energy, as an alternative fuel to gasoline, and it is produced by fermentation methods using plant-derived biomass.
First generation bioethanol is fuel ethanol produced by fermentation using budding yeast or the like, with the starting material being glucose from sugarcane and the like, or glucose obtained by enzymolysis of starch from corn and the like. However, competition of these starting materials with foods and livestock feeds is considered to be a problem.
Second generation bioethanol, on the other hand, is ethanol produced from cellulosic biomass, which does not compete with foods or livestock feeds. Various problems are known to be associated with ethanol production from cellulosic biomass, depending on the combination of the type of starting material, pretreatment method, saccharification process and fermentation process, and solutions to these problems are desired.
Furthermore, while various resources may be considered for cellulosic biomass, ligneous materials are considered especially promising for use because they contain the largest amounts of cellulose, which serves as the starting material for glucose. Ligneous materials also comprise hemicellulose and lignin as main components in addition to cellulose, and the use of hemicellulose, as their second most abundant component after cellulose, has also been an important issue in ethanol production from cellulosic biomass. Hemicellulose is converted to xylose by decomposition with saccharifying enzymes, but since the genes for assimilation of xylose are essentially non-functioning in budding yeast, the issue of extremely low ethanol production efficiency from xylose has been a problem. Therefore, much research is being conducted into introducing xylose metabolizing enzymes of xylose-assimilating organisms into budding yeast, or imparting budding yeast with xylose-assimilating properties by forced expression of endogenous genes associated with xylose metabolism. The enzymes associated with xylose metabolism include xylose reductase, xylitol dehydrogenase, xylulose kinase, xylose isomerase and enzymes involved in the pentose phosphate pathway.
Two different pathways exist for conversion from xylose to xylulose, one being a reductive pathway which is catalyzed by an NADPH-dependent xylose reductase (XR) and a NAD+-dependent xylitol dehydrogenase (XDH), and since the two enzymes have different coenzymes, their imbalance can result in accumulation of xylitol as by-product (NPL 1). In production of ethanol from xylose on an industrial scale, the problem of xylitol accumulation has been linked to reduced ethanol production efficiency, and it is therefore important to find a solution.
The other pathway is catalyzed by xylose isomerase (XI), and is advantageous in that the problem of accumulation of xylitol as a by-product does not occur. A known problem with yeast cells that express xylose isomerase genes is the slow ethanol production rate from xylose. In order to solve these problems, xylose isomerase genes have been cloned from numerous bacteria and fungi, and attempts have been made to express them in yeast cells. It has been so far demonstrated that xylose isomerase genes from Piromyces sp. E2 (NPL 2), Orpinomyces sp. ukk1 (NPL 3), Clostridium phytofermentans ISDg (NPL 4), Ruminococcus flavefaciens 17 (NPL 5), Prevotella ruminicola TC2-24 (NPL 6), Burkholderia cenocepacia J2315 (NPL 7), Clostridium cellulolyticum H10 (NPL 8) and Streptomyces rubiginosus (NPL 9) function in yeast cells and can impart xylose metabolic capacity to host budding yeast, albeit with poor efficiency.
In recent years, high activation of xylose isomerase from Piromyces sp. E2 using molecular evolutionary engineering methods has been reported (NPL 10). In this publication, it is shown that xylose isomerase from mutant Piromyces sp. E2 having mutations introduced at 6 sites (E15D, E114G, E129D, T142S, A177T and V433I) improves ethanol production by increasing the growth rate of budding yeast under aerobic conditions with xylose as the carbon source, as well as their consumption rate of xylose. It is indicated that the xylose isomerase from mutant Piromyces sp. E2 had a Vmax value that was 77% larger than the wild type, while the Km value was approximately twice as high. Among the 6 mutations, E15D and T142S were shown to be the important amino acid mutations for high activation.
It has also been reported that for xylose isomerase from Ruminococcus flavefaciens 17 as well, 5% improvement in activity was achieved by amino acid substitution (G179A) near the xylose bonding site, and 26.8% improvement in activity was achieved by replacing the N-terminal 10 amino acid sequence with the N-terminal 12 amino acid sequence of xylose isomerase from Piromyces sp. E2 (NPL 5).
In regard to xylose isomerase from Streptomyces rubiginosus, it has been reported that several mutant xylose isomerase genes were obtained which had acquired high affinity for xylose, under reaction conditions of pH 6 or lower (NPL 9).
Thus, while much research is being conducted on xylose isomerases for highly efficient bioethanol production from xylose in yeast, the xylose metabolic capacities of yeast in which the xylose isomerase genes have been transferred have each been evaluated under different conditions, and no published reports have dealt with determining which are the biologically-derived xylose isomerase genes for xylose isomerases capable of imparting the most efficient ability to produce bioethanol from xylose, or with comparative examination of different xylose isomerases in the same host strains and under the same expression conditions and culturing conditions. Moreover, while the fermentation conditions that allow the most efficient production of ethanol in budding yeast are anaerobic or microaerobic conditions, most of the reports to date deal with evaluating the functions of xylose isomerases with culturing under aerobic conditions, and not with comparative evaluation under fermentation conditions that allow highly efficient production of ethanol. In addition, even for mutant xylose isomerases that have been highly functionalized by introduction of mutations, it is still unknown whether they contribute to highly efficient bioethanol production under anaerobic or microaerobic conditions.